Vpr from HIV-1 subtypes B and C exhibit significant differences in their ability to transactivate LTR-mediated gene expression and also in their ability to promote apoptotic DNA ladder formation

The ninety-six amino acid long accessory protein Vpr of HIV-1 has been implicated in G2 cell cycle arrest, cytopathogenesis in human T cells and also in augmenting HIV-1 LTR mediated transcription [1]. Most of these functions have been studied using Vpr-B derived from HIV-1 subtype B but comparative data for HIV-1 subtype C is lacking. Among all other genetic subtypes, HIV-1 subtype C is mainly responsible for causing the epidemic in sub-Saharan Africa and some regions of Asia, including India. Since there are significant changes (ten amino acids changes, five of them being conservative and the rest nonconservative) in the amino acids sequence between a prototype subtype B (NL4-3) [2] (Fig. 1) and an Indian isolate of subtype C (clone 93IN905 and accession no. AF067158), it is quite possible that the above mentioned functions could also show variations.Fig. 1: Vpr-B and Vpr-C protein sequence comparison.With this in mind, we cloned the full-length Vpr gene from pNL4-3 and 93IN905 (Indian subtype C isolate) DNA. They were amplified by the polymerase chain reaction (PCR) using the common primers: forward primer: 5′- GC-GGATCC- ATGGAACAAGCCCCAGAAG and reverse primer: 5′-GC- CTCGAG- CTAGGATCTACTGGCTCCATTTC. BamH1 and Xho1 restriction sites were engineered in the forward and reverse primers, respectively, which are indicated in bold. The PCR amplified product (291 bp) was cloned into pcDNA3 expression vector (Promega, Madison, Wisconsin, USA) that was digested with the same two restriction enzymes which placed the Vpr (B and C) gene downstream of the powerful cytomegalovirus immediate–early enhancer/promoter. The authenticity of the recombinant clones was confirmed by sequencing. Recombinant clones were purified on a Qiagen column (Qiagen, GmbH, Hilden, Germany) and used for transfection into mammalian cells for DNA fragmentation studies and HIV-1 LTR-mediated transactivation studies. Briefly, indicated amounts of plasmid DNAs were transfected into mammalian cells (human 293 or Jurkat-T) for 24 h for transactivation studies but for 48 h to study Vpr-mediated DNA fragmentation. To study LTR-mediated transactivation, we employed subtypes B and C specific LTR-reporter gene vectors (pBlue3′-LTR-Luc-B and C) and co-transfected with Vpr encoding vector using Lipofectamine-2000 (Invitrogen, carlsbad, California, USA) as described previously [3]. To ensure uniform transfection efficiency, a control vector (pSV-β-gal) (Promega Biotech.) DNA was always included. LTR-B or C-reporter plasmid DNA was kept constant at 100 ng for 0.5 × 105 Jurkat-T cells in a 24-well plate but the concentration of Vpr encoding plasmid DNAs was varied as indicated in Fig. 2. Lane 1 shows the background reporter gene values obtained with cells only and lane 2 represents cells that received LTR-B-Luciferase construct only along with 100 ng of unrelated plasmid DNA. With increasing amounts of Vpr-B (lane 3, 25 ng; lane 4, 50 ng; lane 5, 100 ng), a dose-dependent increase in the LTR-B-promoter activity was observed. Approximately 20-fold more promoter activity was observed with equivalent amounts (100 ng) of Vpr-B (compare lane 2 with lane 5). When Vpr-C was combined with increasing concentration of LTR-B-Luciferase (lanes 6–8), an approximately four-fold increase was observed compared with lane 2. Lanes 9 to 15 exhibit the promoter activity when a fixed amount of LTR-C-Luciferase (100 ng) but varying amounts of either Vpr-B or -C encoding DNA was added. When LTR-C was combined with Vpr-B, a dose-dependent increase in promoter activity was observed (lanes 9 to 12) compared with LTR-C-Luciferase alone (lane 9). A strong promoter activity was observed in the presence of 100 ng of Vpr-B (lane 12). On the other hand when LTR-C-Luciferase construct was combined with increasing amounts of Vpr-C encoding DNA, only a modest increase (less than two-fold) was observed compared with lane 9. The following important conclusions can be drawn. Vpr-B is more powerful transactivator of LTR-Luciferase promoter than Vpr-C from an Indian isolate in both homologous (LTR-B-Luciferase + Vpr-B) and heterologous system (LTR-C-Luciferase + Vpr-B). Enhanced LTR-C promoter activity in presence of Vpr-B could be of functional relevance in individuals who are co-infected with subtypes B and C. These values comprise the mean ± SD from three independent experiments. An exactly similar pattern of transactivation was observed when the experiment was carried out in HEK-293 cells (data not shown). We then investigated whether Vpr from subtype B and Indian isolate of subtype C differed in their ability to cause DNA fragmentation, which is an indicator of apoptosis. The 293 cells were seeded in 0.5 × 106 cells and transfected with 1 μg of either Vpr-B or -C encoding plasmid DNA in 1 ml volume. DNA was isolated 48 h after transfection and analyzed on a 1.5% agarose gel by ethidium-bromide staining as described previously [4]. Vpr-C was more potent in promoting DNA damage as demonstrated by a more prominent pattern of DNA ladder formation (Fig. 3; compare lanes 2 and 3). The 293 cells, when transfected with the same amounts of empty vector (pcDNA3), as expected, failed to show any DNA fragmentation (lane 1).Fig. 2: Vpr-B and -C mediated transactivation of HIV-1 LTR promoter.Fig. 3: DNA fragmentation by Vpr-B and -C.In summary, we show that Vpr-B was more powerful in activating HIV-1 LTR (B and C) promoter but Vpr-C (Indian isolate) was more potent in causing programmed cell death. These have important implications in modulating HIV-1 subtype-specific pathogenesis. Acknowledgements LTR-Luciferase and pNL4-3 plasmid DNAs were obtained from the AIDS Research & Reference Reagent Program, NIH, Maryland, USA. Sponsorship: This work was supported by a grant from Department of Biotechnology, Government of India, to National Institute of Immunology, JNU Campus, New Delhi, India, and to the corresponding author (A.C.B.).